Stains and fluorescent agents are widely used to visualize structures in cells and tissues. For example, in pathology, it is common to use a hematoxylin and eosin stain preparation (H&E) to visualize structures in cells and tissue. Information obtained from cells stained in this manner can be used to identify cell types, or tissue structures, or to diagnose disease. Other preparations, such as papanicolaou stain, can also be useful for the same broad purpose. These stains are used in brightfield microscopy, where a stained sample is viewed by observing light transmitted through the sample. Absorption or scattering by the stain produces color or contrast in the image of the sample.
DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent counterstain that binds to DNA molecules in nuclei of cells and tissue. It is widely used in fluorescence microscopy to determine the location or presence of nuclei in a sample. In this technique, the sample is excited with light of one wavelength, typically in the 350-370 nm ultraviolet range, and the sample emits light in the violet region of the spectrum. The Hoechst 33258 stain is used for similar purposes, and has excitation and emission properties that are similar to those of DAPI.
Immunofluorescent (IF) probes are useful to image the presence, location, and amount of proteins in cells and tissue. The use of such probes enables highly specific measurements for a variety of research and clinical tasks. Techniques have been developed for applying multiple immunofluorescent probes to a single sample. Optical cross-talk and other factors such as antibody interaction and non-specific binding can limit the number of probes that can be used, and can degrade the accuracy or reduce the useful protein expression range.